WO2024012126A1 - Composé dérivé de cordycépine ayant un effet antitumoral - Google Patents

Composé dérivé de cordycépine ayant un effet antitumoral Download PDF

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WO2024012126A1
WO2024012126A1 PCT/CN2023/099859 CN2023099859W WO2024012126A1 WO 2024012126 A1 WO2024012126 A1 WO 2024012126A1 CN 2023099859 W CN2023099859 W CN 2023099859W WO 2024012126 A1 WO2024012126 A1 WO 2024012126A1
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formula
compound
cordycepin
group
reaction
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PCT/CN2023/099859
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English (en)
Chinese (zh)
Inventor
应汉杰
沈涛
唐成伦
柳东
陈勇
朱晨杰
杨朋朋
庄伟�
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南京工业大学
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Priority to CN202380010911.XA priority Critical patent/CN117321066A/zh
Priority to US18/505,822 priority patent/US20240116976A1/en
Publication of WO2024012126A1 publication Critical patent/WO2024012126A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/167—Purine radicals with ribosyl as the saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/02—Phosphorylation
    • C07H1/04—Introducing polyphosphoric acid radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a derivatized compound based on cordycepin, its preparation method, and its application in the preparation of products for preventing and treating related diseases related to variation in cell functional damage.
  • cancer is a common disease that endangers human life and health.
  • the incidence and mortality of cancer have been on the rise all over the world.
  • the treatment methods for malignant tumors mainly include surgery, radiotherapy and chemotherapy, among which chemotherapy mainly uses synthetic drugs.
  • the inhibitory effect of chemotherapy drugs on tumors is worthy of recognition, and it is also one of the more effective and commonly used treatments for malignant tumors.
  • its toxic and side effects are extensive and serious, and there are drug resistance problems.
  • chemotherapy drugs have poor selectivity between tumor cells and normal cells.
  • nucleosides play an important role in the body's metabolic process. Modification and derivatization of nucleoside compounds is one of the main ways to prepare anti-tumor drugs.
  • nucleoside anti-tumor drugs on the market include forodesine, fludarabine, cladribine, and clofara Bin, fludala phosphate, troxacitabine, etc.
  • Cordycepin (3'-deoxyadenosine) is the main active component of Cordyceps Sinensis. It is a nucleoside analogue. It has effects on cell adaptive changes (anti-aging, anti-cancer), regulating immunity, eliminating inflammation, etc. during the metabolic process in the body. Excellent results.
  • the anti-cancer mechanism of cordycepin is mainly to induce cell apoptosis, regulate cell cycle, and interfere with the expression of matrix metalloproteinases (MMPs), thereby inhibiting tumor cell invasion and metastasis.
  • MMPs matrix metalloproteinases
  • the signaling pathways related to inducing tumor cell apoptosis include the NF- ⁇ B signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway.
  • the main manifestation of regulating the cell cycle is that in cancer cells, cordycepin shortens the G1 phase of the cell cycle, prolongs the G2 phase and M phase, blocks the cell cycle in the G2/M phase, and inhibits cell proliferation.
  • nucleoside analogs have poor lipid solubility, are difficult to absorb, are easily metabolized and inactivated by deaminase, have short half-lives, have low targeting properties, and some tumor cells or viruses are prone to develop drug resistance, etc., which have greatly reduced Effects of using nucleoside drugs.
  • the technical problem to be solved by the present invention is to address the shortcomings of existing nucleoside drugs and to provide a series of chemically modified nucleoside analogues ( cordycepin derivatives).
  • Another technical problem to be solved by the present invention is to provide a composition containing the above-mentioned nucleoside analog.
  • the technical problem to be solved by the present invention is to provide the application of the above-mentioned nucleoside analogues and their compositions in products for preventing and treating related diseases in which functional damage of cells occurs in mammals or humans.
  • the final technical problem to be solved by the present invention is to provide a preparation method for the above-mentioned nucleoside analogues.
  • the present invention discloses a cordycepin derivative represented by formula I, or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrugs, or their metabolites;
  • R 1 is selected from hydrogen, phosphate group, substituted phosphate ester group, phosphonic acid group, substituted phosphonate ester group, alkyl alcohol group, amino acid alkyl ester group, amino acid alkyl alcohol ester group, alkyl acid alkyl ester group ester group, or cycloalkyl polyol group; the substitution is any of an alkoxy group, a halogen-substituted alkoxy group, an aryloxy group, an amino acid ester amide group, an alkyl ester group, or an alkyl acid methyl ester oxygen group.
  • R 2 is selected from hydrogen, or azido
  • R 3 is selected from hydrogen, fluorine, chlorine, or azido
  • R 4 is selected from hydroxyl, cyano, ⁇ -amide- ⁇ -ring Sulfonyloxy group, amino acid carboxylate group, amino acid alkyl ester phosphonate phenyl ester group, or amino acid alkyl ester phosphate phenyl ester group
  • R 5 is selected from hydrogen, bromovinyl group, mercapto group, methyl, fluorine, or chlorine
  • R 6 is selected from amino, substituted carboxamido; the substitution is any one or more functional groups in alkyl, aryl, cycloalkyl, furyl, pyridyl
  • R 7 is selected from hydrogen, or Isopropylamino; and there is no selection of R 1 , R 2 , R 3 , R 5 , R 7 From hydrogen, R 4 is selected from hydroxyl group, and R 6 is selected from
  • R 1 is selected from hydrogen, a phosphate group represented by R 1 1 to R 1 7 , diethyl phosphate, di-n-propyl phosphate, diisopropyl phosphate, diisobutyl phosphate.
  • R 4 is selected from hydroxyl represented by R 4 1 to R 4 7 , cyano, ⁇ -amide- ⁇ -cyclosulfonyloxy, 2-amino-propionyloxy, 2-amino- 3-methyl-butyryloxy, phosphoryloxyalanine methyl ester phenyl ester, or phosphonamidoisoleucine methyl ester phenyl ester; in some embodiments, R 4 is selected from cyano, Cyano group, or a structure represented by any one of R 4 3 , R 4 5 , or R 4 6 .
  • R5 is selected from hydrogen, bromovinyl, thiol, fluorine, or chlorine.
  • R 6 is selected from amino, or acetamido, butylamido, octylamide, dodecylamide, octadecylamide as shown in R 6 1 to R 6 16 , Isopropylamido, isobutylamido, pivalamido, 2-ethyl n-butylamido, 3,3-dimethyl-butylamido, cyclohexylcarboxamide, cyclopentylcarboxamide, benzene carboxamide, furancarboxamide, pyridinecarboxamide, or Hexadecylamide group; in some embodiments, R 6 is selected from amino group, or a structure represented by any one of R 6 5 , R 6 12 , R 6 13 , or R 6 15 .
  • cordycepin derivative shown in formula I or a pharmaceutically acceptable salt thereof; wherein, R 1 is a group represented by R 1 16 , R 2 is selected from hydrogen, and R 3 is selected from Hydrogen, R 4 is selected from hydroxyl, R 5 is selected from hydrogen, fluorine, or chlorine, R 6 is selected from pyridine carboxamide or amino, and R 7 is selected from hydrogen; in some embodiments, cordycepin derived from formula I
  • the compound is selected from compound 24 below, compound 7 below or compound 20 below.
  • the cordycepin derivative represented by Formula I is selected from any one of Compound 1 to Compound 40.
  • the present invention discloses a pharmaceutical composition, which contains at least one of the above-mentioned cordycepin derivatives, or a pharmaceutically acceptable salt, stereoisomer, tautomer thereof Conforms, solvates, prodrugs, or metabolites thereof; and at least one immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is selected from PD-1 and/or CTLA4 monoclonal antibodies.
  • the cordycepin derivative, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, prodrug, or metabolite thereof, and immune checkpoint inhibition The mass ratio of the agent is 1:0.2 ⁇ 10, in some embodiments it is 1:0.2 ⁇ 8, in some embodiments it is 1:0.2 ⁇ 5, in some embodiments it is 1:0.2 ⁇ 3, in some embodiments In some examples, it is 1:0.2 ⁇ 2, in some embodiments it is 1:0.5 ⁇ 1.5, in some embodiments it is 1:0.8 ⁇ 1.2, and in some embodiments it is 1:1.
  • the dosage form of the pharmaceutical composition is selected from tablets, pills, capsules, dropping pills, syrups, disintegrants, injections, sustained-release agents, or kits.
  • the present invention discloses the above-mentioned cordycepin derivatives, or their pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, prodrugs, or their metabolites , or the application of the above-mentioned drug combination in the preparation of products for preventing and treating related diseases in which functional damage of cells occurs in mammals or humans.
  • the disease related to the variation of cell functional damage is a tumor.
  • the tumor includes but is not limited to gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, colon cancer, etc. Rectal cancer, esophageal cancer, prostate cancer, melanoma, glioma, and ovarian cancer; in some embodiments, the tumor is gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, colorectal cancer, melanoma, and ovarian cancer any type of cancer.
  • the products include, but are not limited to, pharmaceuticals.
  • the present invention discloses a preparation method of the above-mentioned cordycepin derivatives.
  • the preparation method is a synthesis method corresponding to the modification method.
  • cordycepin or modified cordycepin molecules Other reactive groups in will participate in the reaction and should be appropriately protected.
  • the protection method involved in the present invention is the protection and deprotection of hydroxyl and amino groups.
  • the protection and deprotection means are conventional means in this field.
  • Some reaction systems require the addition of protective gas for protection, which are common means in experimental processes in this field.
  • R 1 is selected from the group consisting of phosphate group, substituted phosphate ester group, phosphonic acid group, substituted phosphonate ester group, alkyl alcohol group, amino acid alkyl ester group, amino acid alkyl alcohol ester group, alkyl acid Alkyl ester group, or cycloalkyl polyol group; the substitution is alkoxy group, halogen-substituted alkoxy group, aryloxy group, amino acid ester amide group, alkyl ester group, alkyl acid methyl ester oxygen group When any one or more functional groups are substituted; the preparation method of the cordycepin derivative shown in formula I is: using compound IR-1 as a raw material in an organic solvent to undergo a chemical reaction to prepare the cordycepin derivative shown in formula I. ;
  • R 2 to R 7 are the same as R 2 to R 7 in formula I, or are independently selected from protecting groups.
  • the preparation method of the cordycepin derivative shown in Formula I is: in trimethyl phosphite and/or triethyl phosphite, compound IR -1 reacts with the modifier phosphorus oxychloride.
  • the dosage ratio of the compound IR-1, the modifier and the organic solvent is 1 mmol: 3 ⁇ 6 mmol: 15 ⁇ 30 mL.
  • the reaction temperature is -10 ⁇ 5°C, and in some embodiments, the reaction time is 0.5 ⁇ 2h.
  • the preparation method of the cordycepin derivative shown in Formula I is: in anhydrous N,N-dimethylformamide and/or In tetrahydrofuran, using tert-butyl magnesium chloride as a catalyst, compound IR-1 reacts with modifier-substituted nitrophenyl phosphate, the substitution is alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide group, substituted by any one or more functional groups of alkyl ester group and alkyl acid methyl esteroxy group.
  • the compound IR-1, modified The dosage ratio of agent, catalyst and organic solvent is 1mmol: 1 ⁇ 3mmol: 1 ⁇ 2mmol: 9 ⁇ 15mL.
  • the temperature of the reaction is 20 ⁇ 40°C.
  • the reaction The time is 2 ⁇ 5h.
  • the preparation method of the cordycepin derivative shown in Formula I is: in anhydrous N,N-dimethyl In the base formamide, using NaH as the catalyst, compound IR-1 reacts with the modifier substituted or unsubstituted p-toluenesulfonyloxymethylphosphate, the substitution is the alkoxy group or the halogen-substituted alkane.
  • Oxygen group, aryloxy group, amino acid ester amide group, alkyl ester group, alkyl acid methyl ester oxygen group is substituted by any one or more functional groups.
  • the compound IR-1, modifier , the usage ratio of catalyst and organic solvent is 0.1mol: 0.1 ⁇ 0.15mol: 0.2 ⁇ 0.03mol: 100 ⁇ 200mL, in some embodiments it is 0.1mol: 0.1 ⁇ 0.15mol: 0.2 ⁇ 0.03mol: 150mL, in some embodiments
  • the reaction temperature is -20 ⁇ 0°C, and in some embodiments, the reaction time is 0.5-6h.
  • R1 in Formula I is selected from an alkyl alcohol group, an amino acid alkyl ester group, an amino acid alkyl alcohol ester group, an alkyl acid alkyl ester group, or a cycloalkyl polyol group
  • the preparation method of the cordycepin derivative shown in formula I is: in methyl ethyl ketone, using potassium carbonate as a catalyst, compound IR-1 reacts with a modifier, and the modifier is an alkyl alcohol corresponding to a halogen substitution, an amino acid alkyl alcohol, ester, amino acid alkyl alcohol ester, alkyl acid alkyl ester or cycloalkyl polyol, in some embodiments, the halogen is bromine or chlorine, in some embodiments, the compound IR-1, modified
  • the usage ratio of sexual agent, catalyst and organic solvent is 1mmol: 0.5 ⁇ 1.5mmol: 1 ⁇ 3mmol: 5 ⁇ 8mL. In some embodiments, it is 1mmol: 1mmol: 1 ⁇ 3
  • R 2 is selected from an azido group
  • the preparation method of the cordycepin derivative shown in formula I is: using compound IR-2 as a raw material to perform a cyclization reaction in an organic solvent to prepare the intermediate IR- 2a, 5-(6-amino-9H-purin-9-yl)-1,4-dioxapyrrole[2.4]heptan-6-ol or its derivatives; in organic solvents, the obtained intermediate IR- 2a undergoes a ring-opening reaction to obtain the cordycepin derivative represented by Formula I;
  • R 1 and R 3 to R 7 are the same as R 1 and R 3 to R 7 in formula I, or they are independently selected from protecting groups.
  • the preparation method of the intermediate I-R-2a is: in dichloromethane, compound I-R-2 is catalyzed by phosphorus pentoxide and meta-chloroperoxybenzoic acid to perform a cyclization reaction.
  • the usage ratio of the compound I-R-2, phosphorus pentoxide, m-chloroperoxybenzoic acid and methylene chloride is 1mmol: 1.2 ⁇ 2mmol: 2 ⁇ 3mmol: 10 ⁇ 20mL.
  • the temperature of the cyclization reaction is 20-60°C, and in some embodiments, the time of the cyclization reaction is 3-10 hours.
  • the preparation method of the cordycepin derivative shown in formula I is: in anhydrous dimethylformamide, the obtained intermediate I-R-2a is subjected to a ring-opening reaction with sodium azide.
  • the usage ratio of compound I-R-2a, sodium azide and dimethylformamide is 1 mmol: 4-5 mmol: 2-5 mL.
  • the temperature of the ring-opening reaction is 100-100 °C. 120°C.
  • the ring-opening reaction time is 12 to 16 hours.
  • R 3 is selected from fluorine, chlorine, or azide group;
  • the preparation method of the cordycepin derivative shown in formula I is: in an organic solvent, using compound IR-3 as raw material through chemical reaction to prepare Cordycepin derivatives represented by formula I;
  • R 1 , R 2 , R 5 to R 7 are the same as R 1 , R 2 , R 5 to R 7 in formula I, or are independently selected from protecting groups.
  • the preparation method of the cordycepin derivative shown in formula I is: in pyridine and dichloromethane, compound IR-3 and trifluoromethyl Sulfonic anhydride reaction produces intermediate IR-3a, 5-(6-amino-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl trifluoromethanesulfonate Or its derivatives; in ethyl acetate, intermediate IR-3a is subjected to a substitution reaction with hydrofluoric acid, sulfur trifluoride or hydrochloric acid to prepare the cordycepin derivative represented by formula I; wherein, the intermediate In the preparation method of IR-3a, in some embodiments, the usage ratio of the compound IR-3, trifluoromethanesulfonic anhydride, pyridine and dichloromethane is 1mmol: 1 ⁇ 1.5mmol: 0.15 ⁇ 0.2mL: 10 ⁇ 20
  • the preparation method of the cordycepin derivative shown in formula I is: in N,N-dimethylformamide, compound IR-3 is added in The cyclization reaction is carried out under the catalysis of triphenylphosphine and diisopropyl azodicarboxylate to prepare the intermediate IR-3b, 4-(6-amino-9H-purin-9-yl)-3,6-dioxo Heterocycle [3.1.0]hexan-2-yl) methanol or its derivatives; in dimethylformamide, the obtained intermediate IR-3b is reacted with sodium azide through ring-opening reaction to obtain formula I Cordycepin derivatives; wherein, in the preparation method of the intermediate IR-3b, in some embodiments, the compound IR-3, triphenylphosphorus, diisopropyl azodicarboxylate and N,N -The usage ratio of dimethylformamide is 10.0mmol:16
  • R 4 is selected from cyano group, ⁇ -amide- ⁇ -cyclosulfonyloxy group, amino acid carboxylate group, amino acid alkyl ester phosphonate phenyl ester group, or amino acid alkyl ester phosphate phenyl ester group ;
  • the preparation method of the cordycepin derivative represented by formula I is: using compound IR-4 as a raw material in an organic solvent to undergo a chemical reaction to prepare the cordycepin derivative represented by formula I;
  • R 1 to R 3 and R 5 to R 7 are the same as R 1 to R 3 and R 5 to R 7 in formula I, or they are independently selected from protecting groups.
  • the preparation method of the cordycepin derivative shown in Formula I is: in dichloromethane, first combine compound IR-4, trifluoromethanesulfonic acid, Trimethylsilyl trifluoromethanesulfonate is stirred in an environment of -50 to -30°C, and then reacted with trimethylsilyl nitrile and triethylamine; in some embodiments, the compound IR-4, trifluoromethanesulfonate Acid, trimethylsilyl trifluoromethanesulfonate
  • the usage ratio of ester and methylene chloride is 10mmol: 0.8 ⁇ 1.8mL: 2.2 ⁇ 3.2mL: 90 ⁇ 110mL.
  • the stirring The time is 20 ⁇ 40min, in some embodiments it is 30min; in some embodiments, the dosage ratio of the compound IR-4 to trimethylnitrile silane and triethylamine is 10mmol: 3.4 ⁇ 4.3g: 3 ⁇ 4 mL.
  • the reaction temperature is 20-30°C, and in some embodiments it is room temperature. In some embodiments, the reaction time is 2-4 hours.
  • the preparation method of the cordycepin derivative shown in formula I is: compound IR-4 is first subjected to an oxidation reaction to prepare the intermediate IR-4a, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)dihydrofuran-3(2H)-one or its derivatives, The intermediate IR-4b is then prepared by cyanolation reaction, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)-3-isocyanotetrahydrofuran- 3-alcohol or its derivatives are then esterified with methanesulfonate to prepare intermediate IR-4c, 2-(6-amino-1,6-dihydro-9H-purin-9-yl)-5-(hydroxy Methyl)-3-isocyanotetra
  • the dosage ratio of compound IR-4, Jones reagent and acetone is 20 mmol: 5 ⁇ 8mL: 50 ⁇ 100mL.
  • the reaction temperature is 0 ⁇ 40°C.
  • the reaction time is 1 ⁇ 4h; in some embodiments, the intermediate IR
  • the preparation method of -4b is to react intermediate IR-4a with trimethylsilyl cyanide and boron trifluoride ether in dichloromethane.
  • the intermediate IR-4a trimethylsilyl cyanide,
  • the usage ratio of silicon cyanide, boron trifluoride ether and methylene chloride is 20mmol: 20 ⁇ 40mmol: 10 ⁇ 30mmol: 50 ⁇ 100mL, in some embodiments it is 20mmol: 20 ⁇ 40mmol: 20mmol: 50 ⁇ 100mL, in some embodiments, the reaction temperature is 0-40°C, and in some embodiments, the reaction time is 1-4 hours; in some embodiments, the preparation method of the intermediate IR-4c is: In anhydrous methylene chloride, intermediate IR-4b is reacted with triethylamine and methanesulfonyl chloride.
  • the intermediate IR-4b, triethylamine, methanesulfonyl chloride and anhydrous methylene chloride are reacted.
  • the dosage is 2.2mmol: 10 ⁇ 15mmol: 4 ⁇ 8mmol: 5 ⁇ 10mL.
  • the reaction temperature is -30 ⁇ 0°C.
  • the reaction time is 2 ⁇ 4h;
  • the preparation method of the cordycepin derivative described in Formula I is: reacting intermediate IR-4c with cesium carbonate in anhydrous acetonitrile.
  • the intermediate The usage ratio of IR-4c, cesium carbonate and anhydrous acetonitrile is 1mmol: 0.5 ⁇ 2.5mmol: 3 ⁇ 10mL, in some embodiments it is 1mmol: 1.5mmol: 3 ⁇ 10mL.
  • the reaction The temperature ranges from 0 to 40°C. In some embodiments, the reaction time ranges from 2 to 4 hours.
  • the preparation method of the cordycepin derivative shown in Formula I is: reacting compound IR-4 with an amino acid chloride in anhydrous pyridine, The cordycepin derivative shown in formula I is prepared; in some embodiments, the dosage ratio of the compound IR-4, amino acid chloride and pyridine is 10mmol: 5 ⁇ 15mmol: 50 ⁇ 100mL, in some embodiments it is 10mmol : 10 mmol: 50 to 100 mL.
  • the reaction temperature is 20 to 60°C, and in some embodiments is 40°C.
  • the reaction time is 6 to 20 h.
  • the preparation method of the cordycepin derivative shown in Formula I is: in anhydrous N,N-dimethylformamide In, using NaH as a catalyst, compound IR-4 reacts with p-toluenesulfonyloxymethylphenyl phosphate substituted by the modifier amino acid alkyl ester.
  • the compound IR-4, the modifier, The usage ratio of NaH and anhydrous N,N-dimethylformamide is 0.1mol:0.1 ⁇ 0.15mol:0.2 ⁇ 0.03mol:100 ⁇ 200mL, in some embodiments it is 0.1mol:0.1 ⁇ 0.15mol:0.2 ⁇ 0.03 mol: 150 mL.
  • the reaction temperature is -20 ⁇ 0°C.
  • the reaction time is 0.5-6h.
  • the preparation method of the cordycepin derivative shown in Formula I is: in anhydrous N,N-dimethylformamide and /or in tetrahydrofuran, using tert-butyl magnesium chloride as the catalyst, compound IR-4 reacts with nitrophenyl phosphate substituted with a modifier, the substitution is any one of aryloxy group and alkyl acid methyl ester oxygen group Or multiple functional groups are substituted.
  • the usage ratio of the compound IR-4, modifier, catalyst and organic solvent is 1mmol: 1 ⁇ 3mmol: 1 ⁇ 2mmol: 9 ⁇ 15mL.
  • the reaction temperature is 20-40°C, and in some embodiments, the reaction time is 2-5 hours.
  • R 5 is selected from bromovinyl group, mercapto group, methyl, fluorine, or chlorine; the preparation method of the cordycepin derivative shown in formula I is: using compound IR-5 as raw material in an organic solvent through chemical Reaction to prepare the cordycepin derivative represented by formula I;
  • R 1 to R 4 and R 6 to R 7 are the same as R 1 to R 4 and R 6 to R 7 in formula I, or they are independently selected from protecting groups.
  • the preparation method of the cordycepin derivative shown in formula I is: subjecting compound IR-5 to iodination reaction to prepare intermediate IR-5a, 2 -(6-Amino-2-iodo-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-ol or its derivatives, intermediate IR-5a via acrylic acid Methyl ester substitution reaction produces intermediate IR-5b, methyl (E)-3-(6-amino-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6,9-dihydro- 1H-Purin-2-yl)acrylate or its derivatives, intermediate IR-5b is hydrolyzed to obtain intermediate IR-5c, (E)-3-(6-amino-9-3-hydroxy-5-( Hydroxymethyl)tetrahydrofuran-2-yl)-6,9-di
  • the usage ratio of the compound IR-5 to iodine element is 1 mmol: 0.5-0.8 mmol.
  • the reaction temperature is 100-120°C. In some embodiments, the reaction temperature is 100-120°C. In the embodiments, it is 110°C. In some embodiments, the reaction time is 4 to 6 hours; in some embodiments, the preparation method of the intermediate IR-5b is: in 1,4-dioxane In, intermediate IR-5a reacts with methyl acrylate and triethylamine under the catalysis of palladium acetate and triphenylphosphine.
  • the intermediate IR-5a, methyl acrylate, triethylamine The usage ratio of palladium acetate, triphenylphosphine and 1,4-dioxane is 1mmol: 3 ⁇ 4mmol: 0.1 ⁇ 0.5mL: 0.01 ⁇ 0.09mmol: 0.05 ⁇ 0.15mmol: 10 ⁇ 20mL, in some embodiments It is 1mmol: 3 ⁇ 4mmol: 0.1 ⁇ 0.5mL: 0.05mmol: 0.01mmol: 10 ⁇ 20mL.
  • the reaction temperature is 50 ⁇ 90°C.
  • the reaction time is 0.5 ⁇ 2h; in some embodiments, the preparation method of the intermediate IR-5c is to perform a hydrolysis reaction between the intermediate IR-5b and a sodium hydroxide solution.
  • the sodium hydroxide solution is The concentration is 0.5 ⁇ 3.5mol/L, in some embodiments it is 2mol/L.
  • the dosage ratio of the intermediate IR-5b to the sodium hydroxide solution is 1g:10 ⁇ 14mL, in some embodiments In the example, it is 1g:12mL.
  • the reaction temperature is 20-30°C, and in some embodiments it is room temperature.
  • the reaction time is 3-5 hours; in some implementations
  • the preparation method of the cordycepin derivative described in formula I is as follows: in a mixed solvent of water and acetone, the intermediate IR-5c and N-bromosuccinimide are reacted under the catalysis of potassium carbonate.
  • the usage ratio of the intermediate IR-5c, N-bromosuccinimide, potassium carbonate, and mixed solvent is 1 mmol: 1 to 3 mmol: 1 to 3 mmol: 15 to 30 mL.
  • the The volume ratio of water to acetone is 1:4-8, in some embodiments it is 1:6.
  • unmodified cordycepin is directly modified with bromovinyl, and the reaction path is as follows:
  • the preparation method of the cordycepin derivative shown in Formula I is: reacting compound IR-5 with hydrogen peroxide in acetic acid to prepare intermediate IR-5d , 1N-oxidized-3'-deoxyadenosine or its derivatives, the intermediate IR-5d is heated and refluxed in a hydrochloric acid aqueous solution to prepare the intermediate IR-5e, 5-amino-N'-hydroxy-3-hydroxy- 5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-imidazole-4-carboxamide or its derivatives, the intermediate IR-5e is dissolved in water, catalyzed by Raney nickel, and reacted in a hydrogen environment to prepare Obtain intermediate IR-5f, 5-amino-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-imidazole-4-carboxamide or its derivatives, and combine intermediate IR-5f with methanol
  • the dosage ratio of compound IR-5, hydrogen peroxide and acetic acid is 1 mmol: 2 to 20%. 3 mol: 1 to 6 mL.
  • the reaction temperature is 30 to 50°C.
  • the reaction time is 2 to 4 days; in some embodiments, the intermediate The preparation method of intermediate IR-5e is to heat and reflux intermediate IR-5d in an aqueous hydrochloric acid solution.
  • the concentration of the aqueous hydrochloric acid solution is 1 to 5 mol/L, and in some embodiments it is 3 mol/L.
  • the dosage ratio of the intermediate IR-5d and the hydrochloric acid aqueous solution is 1 mmol: 3 to 5 mL.
  • the heating reflux time is 10 to 30 min; wherein, the intermediate In the preparation method of the intermediate IR-5f, in some embodiments, the usage ratio of the intermediate IR-5e, Raney nickel and water is 1mmol:0.08 ⁇ 0.2g:10 ⁇ 20mL.
  • the The temperature of the reaction is 50-70°C, and in some embodiments, the reaction time is 2-5 days; wherein, in the preparation method of the cordycepin derivative described in Formula I, in some embodiments, The usage ratio of intermediate IR-5f to methanol, pyridine and carbon disulfide is 1 mmol: 5 to 10 mL. In some embodiments, the volume ratio of methanol, pyridine and carbon disulfide is 4: 3 to 7: 0.5 to 3.5. In some embodiments, it is 4:5:2. In some embodiments, the reaction temperature is 30-50°C, and in some embodiments it is 40°C. In some embodiments, the reaction time is 3 ⁇ 5 days.
  • sulfhydryl modification is performed directly on unmodified cordycepin.
  • the specific reaction method is as follows:
  • the preparation method of the cordycepin derivative shown in formula I is: reacting compound IR-5, methyl iodide and potassium carbonate in dichloromethane solvent,
  • the usage ratio of the compound IR-5, methyl iodide, potassium carbonate and dichloromethane is 1 mmol: 1-2 mmol: 1.5-3 mmol: 5-10 mL.
  • the reaction The temperature is 20-50°C.
  • the reaction time is 3-10 h.
  • the preparation method of the cordycepin derivative shown in formula I is: subjecting compound IR-5 to a nitro derivatization reaction to prepare a nitrolation intermediate IR-5g, 2-(6-amino-2-nitro-1,6-dihydro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-ol, and then carry out substitution reaction,
  • the cordycepin derivative shown in formula I is prepared; in some embodiments, the preparation method of the intermediate IR-5g is to combine compound IR-5 and tetrabutylammonium nitrate in trifluoromethane.
  • the reaction is carried out under the catalysis of acetic anhydride.
  • the dosage ratio of the compound IR-5, tetrabutylammonium nitrate, trifluoroacetic acid and dichloromethane is 1mmol: 1.4 ⁇ 2mmol: 1 ⁇ 2mmol: 15 ⁇ 35mL , in some embodiments, the temperature of the reaction is -10 ⁇ 10°C, in some embodiments, the time of the reaction is 0.5 ⁇ 20h; in some embodiments, the preparation of the cordycepin derivatives described in Formula I
  • the method is to react the nitrated intermediate IR-5g with tetrabutylammonium fluoride or tetrabutylammonium chloride in acetonitrile.
  • the nitrated intermediate IR-5g The dosage ratio of tetrabutylammonium fluoride or tetrabutylammonium chloride to acetonitrile is 1 mmol: 1.3 ⁇ 1.5 mmol: 30 ⁇ 50 mL, and the temperature of the reaction is -5 ⁇ 5°C, in some embodiments it is 0°C , in some embodiments, the reaction time is 20 to 30 minutes.
  • R 6 is selected from a substituted carboxamido group; when the substitution is any one or more functional groups among alkyl, aryl, cycloalkyl, furyl, and pyridyl; as shown in formula I
  • the preparation method of cordycepin derivatives is as follows: using compound IR-6 as a raw material through chemical reaction in an organic solvent to prepare the cordycepin derivative represented by formula I;
  • R 1 to R 5 and R 7 are the same as R 1 to R 5 and R 7 in formula I, or they are independently selected from protecting groups.
  • the preparation method of the cordycepin derivative shown in formula I is: reacting compound I-R-6 with a substituted acid chloride under anhydrous pyridine, and the substitution is an alkyl group, an aryl group, or a cycloalkyl group. , furyl, pyridyl, any one or more functional groups substituted.
  • the dosage ratio of compound I-R-6, substituted acid chloride and anhydrous pyridine is 1mmol: 1 ⁇ 2mmol: 5 ⁇ 10mL, in some embodiments
  • the reaction temperature is 0-60°C, and in some embodiments, the reaction time is 2-20 h.
  • R 7 is selected from isopropylamino;
  • the preparation method of the cordycepin derivative shown in formula I is: using compound IR-7 as a raw material in an organic solvent through chemical reaction to prepare formula I Cordycepin derivatives;
  • R 1 to R 6 are the same as R 1 to R 6 in formula I, or are independently selected from protecting groups.
  • the preparation method of the cordycepin derivative shown in formula I is: in dioxane, compound I-R-7 is substituted with 2-propylamine, and in some embodiments, compound I-R-7 is reacted with The dosage ratio of 2-propylamine and dioxane is 10mmol: 10 ⁇ 30mmol: 50 ⁇ 100mL. In some embodiments, the reaction temperature is 60 ⁇ 120°C. In some embodiments, the reaction time It is 10 ⁇ 30h.
  • the protecting group includes but is not limited to -OTBS, -OAc, -NHCbz, -OTBPS, -OTBDPS; in some embodiments, R 4 is the same as R 4 in formula I or is selected from -OTBS, -OAc , -OTBDPS; in some embodiments, R 6 is the same as R 6 in formula I or selected from -NHCbz.
  • the substituted nitrophenyl phosphate (the substitution is an alkoxy group, a halogen-substituted alkoxy group, an aryloxy group, an amino acid ester amide group, an alkyl ester group, an alkyl acid methyl ester oxygen group substituted by any one or more functional groups) can be prepared according to the following method or other methods in the prior art.
  • phenyl chlorophosphate, p-nitrophenol and corresponding substituted alcohols or amines are prepared under the catalysis of triethylamine in an environment of 0 to 25°C.
  • the substitutions are alkoxy, halogen Substituted with any one or more functional groups in a substituted alkoxy group, aryloxy group, amino acid ester amide group, alkyl ester group, and alkyl acid methyl ester oxygen group.
  • the phenyl chlorophosphate The molar volume ratio of p-nitrophenol, corresponding substituent, triethylamine and anhydrous dichloromethane is 1mmol: 1mmol: 1 ⁇ 2mmol: 2 ⁇ 5mmol: 5 ⁇ 10mL.
  • the substituted p-toluenesulfonyloxymethylphosphate (the substitution is alkoxy, halogen-substituted alkoxy, aryloxy, amino acid ester amide group, alkyl ester group, alkyl acid (substituted by any one or more functional groups in the methyl ester oxygen group) can be prepared according to the following method or other methods in the prior art.
  • chlorophosphates Correspondingly substituted chlorophosphates, p-toluenesulfonyl chloride, and formaldehyde in toluene are prepared through triethylamine catalysis in an environment of 0 to 105°C.
  • the substituted chlorophosphates are alkoxy groups or halogen-substituted alkoxy groups.
  • the corresponding substituted chlorophosphate ester the molar volume ratio of p-toluenesulfonyl chloride, formaldehyde, triethylamine and toluene is 1mol: 1mol: 0.8 ⁇ 1.2mol: 180 ⁇ 210mL: 500 ⁇ 800mL.
  • the present invention is based on cordycepin to modify R 1 to R 7.
  • R 1 is hydrogen
  • R 2 is hydrogen
  • R 3 is hydrogen
  • R 5 is hydrogen
  • R 7 When R 4 is hydrogen, R 4 is hydroxyl, or R 6 is amino, there is no need to modify it.
  • the above-mentioned substitutions can be modified according to the priority of the stability of the prepared intermediates without violating common sense in the field, that is, each example of the present invention can be obtained.
  • the intermediates can be modified first. Modifications that do not contain ester groups, phosphate groups, or phosphonic acid groups, such as -CN, -N 3 , -F, -SH, etc. modifications, then ester group modifications, and finally phosphate or phosphonic acid group modifications .
  • prevention means the administration of a compound or formulation described herein to prevent a disease or one or more symptoms associated with said disease, and includes: preventing the occurrence of a disease or disease state in a mammal, Especially when these mammals are prone to induce related cancer symptoms.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that are suitable for use in contact with human and animal tissues within the scope of reliable medical judgment. without undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers isomer, the (D)-isomer, the (L)-isomer, as well as their racemic mixtures and other mixtures, such as enantiomeric or diastereomerically enriched mixtures, all of which belong to the present invention. within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • the present invention has the following advantages:
  • cordycepin derivatives and pharmaceutical compositions thereof provided by the present invention have good anti-tumor proliferation effects.
  • cordycepin derivatives Compared with the parent drug, cordycepin derivatives have better affinity for cell membranes, allowing the drug to have a longer half-life of metabolism in the body and remain in the body for a longer time.
  • the cordycepin derivatives and their pharmaceutical compositions provided by the present invention have a wider range of types and effects on tumors, including gastric cancer, pancreatic cancer, liver cancer, small cell lung cancer, colorectal cancer, and gastric cancer. Cancer, melanoma, ovarian cancer, etc. all have excellent inhibitory effects, lower side effects, and better efficacy.
  • Figure 1 shows the anti-tumor effects of the blank group, the cordycepin control group and the compound group on the liver cancer cell Hep-1-6 transplanted mouse model.
  • Figure 2 shows the inhibitory effects of the control group and each compound group on the small cell lung cancer H446 cell tumor-bearing zebrafish model.
  • Figure 3 shows the inhibitory effect of compound 16 and immune checkpoint inhibitors on the colon cancer MC38 cell transplantation mouse model.
  • Figure 4 shows the anti-tumor effects of the control group and the compound group on the melanoma B16-F10 transplanted mouse model.
  • Figure 5 shows the inhibitory effect of compound 24 and immune checkpoint inhibitors on the mouse ID8 ovarian cancer homograft tumor model.
  • Figure 6 shows the anti-tumor effects of the control group and the compound group on the gastric cancer BGC-823 cell transplantation mouse model.
  • Figure 7 shows the anti-tumor effects of the control group and the compound group on the pancreatic cancer Pan02-luc cell transplanted mouse model.
  • the cordycepin described in the present invention is derived from the preparation of biological fermentation pathway (CN 111117896B), wherein the preparation method of cordycepin or its derivatives is prepared according to the method in the instructions, and the post-processing method mainly includes but is not limited to filtration, quenching Conventional organic experimental post-processing operations such as sterilization, extraction, rotary evaporation, recrystallization, column chromatography, etc.
  • the preparation methods of the compounds mentioned in the examples are not all compounds. For convenience of explanation, only the preparation processes of several representative compounds are listed here.
  • the anti-cancer related cell and animal model experiments of cordycepin or its derivatives are not all effective experimental results, but are only related experiments listed to illustrate the effects, and the drug evaluation experiments include cell models and animal models. Experiments on models follow ethical rules.
  • mice are raised and treated in a laminar flow cabinet. 5 nude mice are raised in each cage, drinking water and feed are added once every 3 days, and the bedding is changed once a week; The number of mice should not exceed 5. Mice in separate cages should be allowed to live in groups as much as possible, and mice should not be placed in single cages. Animal management must comply with relevant national standards for animal feeding and management. At the same time, attention should be paid to the behavioral needs of the animals to avoid undesirable consequences.
  • Necessary stress meet the normal physiological and behavioral needs of animals, such as defecation, urination, maintaining a constant body temperature, normal activities, adjusting posture and breeding; good ventilation to keep animals dry; free access to drinking water and food, and easy to supplement and Replacement operations and cleaning; provide a strong and safe environment to avoid accidents such as animals escaping or limbs getting stuck in gaps; avoid harm to animals from sharp edges or protrusions; and avoid disturbing animals when observing animals.
  • the zebrafish model described therein is wild-type AB strain zebrafish, which comes from the School of Biological and Pharmaceutical Engineering of Nanjing University of Technology. It is reproduced in a natural pair-mating breeding method. There are 30 animals in each experimental group, and the age is 2dpf. Raise in fish farming water at 28°C (water quality: add 200mg instant sea salt per 1L reverse osmosis water, conductivity 480 ⁇ 510uS/cm; pH 6.9 ⁇ 7.2; hardness 53.7 ⁇ 71.6mg/L CaCO 3 ). Management meets the requirements of international AAALAC certification.
  • the MTT method described therein determines the inhibitory activity of compounds on tumor cell proliferation as follows: Take a bottle of cells in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, and count 2 to 4 ⁇ 10 4 cells/mL to make a cell suspension. Take the cell suspension and inoculate it on a 96-well plate, 90 ⁇ L/well, and culture it in a constant-temperature CO2 incubator for 24 hours. Add the prepared compound for testing, 10 ⁇ L/well, and incubate for 72 hours. Add MTT reagent to the 96-well plate, 10 ⁇ L/well, and react in the incubator for 4 hours.
  • Example 1 Preparation of compound: 2-(6-amino-9H-purin-9-yl)-5-(2-hydroxyethoxy)methyl)tetrahydrofuran-3-ol;
  • the detection results of the prepared compound 1 are as follows, 1 H NMR (400MHz, DMSO-d6) ⁇ 8.56 (s, 1H), 8.35 (s, 1H), 7.09 (s, 2H), 6.14 (d, 1H), 5.47-5.3(d,2H),5.01(m,1H),4.12(m,1H),3.74(m,1H),3.61-3.56(m,4H),3.51-3.46(m,2H),2.04- 1.92(m,2H).
  • Example 1 According to the protection method in Example 1, take 3.65g (10mmol) of compound 1b and add 150mL of triethyl phosphite and 4.62g (30mmol) of phosphorus oxychloride under ice bath conditions. The reaction was carried out for 2 hours at 0°C. After the reaction was completed, 1000 mL of water was added in an ice bath environment to quench the reaction. Dichloromethane was added for multiple extractions. The organic phases were combined and the solvent was removed by rotary evaporation to obtain a viscous liquid.
  • Compound 2 was prepared according to the deprotection scheme of 1b in Example 1.
  • the detection results of the prepared compound 2 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.54 (s, 1H), 8.36 (s, 1H),7.11(s,2H),6.19(d,1H),5.15(d,1H),4.28-4.23(m,2H),4.2(s,2H),4.02(m,1H),3.71(m ,1H),2.06-1.90(m,2H).
  • reaction solution is filtered and washed with dichloromethane.
  • the filtrate is extracted three times with water (60 mL).
  • the dichloromethane phases are combined and the solvent is evaporated under reduced pressure.
  • Toluene was added to the residue and recrystallized.
  • the crystallization mother liquor was filtered, and the filter cake was dried under reduced pressure at 50°C to obtain a white powdery solid 3a, which was weighed to obtain 26.87g, with a yield of 52%, MSI-MS: 540.6[M+23] + .
  • Compound 5 was prepared according to the method for preparing 3a in Example 3 and combined with the deprotection method of 1b in Example 1, in which equivalent amounts of p-toluenesulfonyloxymethyl diethyl phosphate were replaced by (((tolyloxy)methyl )phosphoryl)bis(oxy))bis(methylene)bis(2-methylpropionic acid), the overall yield of compound 5 was 74%.
  • the detection results of the prepared compound 5 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.57 (s, 1H), 8.33 (s, 1H), 7.11 (s, 2H), 6.84 (d, 4H), 6.18(d,1H),5.46(d,1H),4.12(m,1H),3.94(m,1H),3.81(d,2H),3.60-3.44(m,2H),2.55(m,2H) ,2.08-1.82(m,2H),1.14(d,12H).
  • Example 6 Preparation of compound: Methyl ((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-D -Valine, wherein the preparation process is as follows:
  • Methyl((4-nitrophenoxy)(phenoxy)phosphoryl)-D-valine is prepared as follows:
  • the detection results of the finally prepared compound 6 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.58(s,1H),8.33(s,1H),7.43(m,2H),7.20(m,3H)7.08 (s,2H),6.15(d,1H),5.47(d,1H),4.26-4.12(m,2H),4.02(m,1H),3.75(m,1H),3.67(d,1H), 3.62(s,3H),3.28(d,1H),2.41(m,1H),2.08-1.96(m,2H),0.98(d,6H).
  • Example 7 Preparation of compound: isopropyl ((((((((((((((((((((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl )(phenoxy)phosphoryl)alanine ester, the preparation process is as follows:
  • the reaction was cooled to room temperature, 80 mL of saturated sodium bicarbonate aqueous solution was slowly added, and then sodium bicarbonate solid powder was added to adjust the pH value of the reaction solution to 7-8. Filter to remove the precipitated salt, place the filtrate in a separatory funnel to separate the chloroform layer, extract the aqueous layer once with 100 mL of chloroform, combine the organic phases, wash once with saturated sodium bicarbonate aqueous solution and water in turn, take the chloroform layer and decompress After distillation, the residue is the target product phenyl chlorophosphate alanine isopropyl ester, with a yield of 87%.
  • Compound 7 was prepared according to the method for preparing 3a in Example 3 and combined with the deprotection method of 1b in Example 1, in which equivalent amounts of p-toluenesulfonyloxymethylphosphate diethyl ester was replaced with isopropyl (phenoxy (( Tolyloxy)methyl)phosphoryl)alanine ester, compound 7 was prepared in 52% yield.
  • the detection results of the prepared compound 7 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.59 (s, 1H), 8.37 (s, 1H), 7.45 (m, 2H), 7.24 (m, 3H), 7.12(s,2H),6.16(d,1H),5.33(d,1H),4.98(m,1H),4.08-3.99(m,2H),3.84(m,1H),3.77(m,2H) ,3.58-3.42(m,3H),2.08-1.96(m,2H),1.29(d,3H),1.16(d,6H).
  • compound 8 was prepared according to the deprotection method of 1b in Example 1, with a yield of 91%.
  • the detection results of the prepared compound 8 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.88(s,2H),8.54(s,1H),8.37(s,1H),7.14(s,2H), 6.18(d,1H),5.57(d,1H),4.33-4.11(m,3H)4.03(m,1H),3.96-3.89(m,3H),3.61-3.36(m,4H),2.37(m ,1H),2.08-1.82(m,2H),0.98(d,6H).
  • Example 9 Preparation of compound: (4-amino-6-(6-amino-9H-purin-9-yl)-2,2-dioxide-1,7-dioxa-2-thispirocycle [ 4.4] Non-3-en-8-yl) dihydrogen methyl phosphate, the preparation method is as follows:
  • the detection results of the prepared compound 9 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.59(s,1H),8.38(s,1H),7.14(s,2H),6.88(s,2H), 6.16(d,1H),5.25(s,1H),4.28(m,1H),4.18(s,2H),4.02(m,1H),3.74(m,1H),2.09-1.96(m,2H) . 13 C NMR (100MHz, DMSO-d6) ⁇ 168.5,155.1,151.4,147.8,141.1,119.4,98.2,88.9,86.1,73.0,68.1,37.9.MSI-MS:435.3[M+H] + .
  • protection and deprotection may be performed by referring to the preparation method of 1a in Example 1, wherein the protected 1a undergoes an acylation reaction in the second step, and then is deprotected to prepare compound 10.
  • the detection results of the prepared compound 10 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 10.55(s,1H),8.64(s,1H),8.38(s,1H),6.18(d,1H), 5.35(d,1H),5.04(m,1H),4.02(m,1H),3.76(m,1H),3.58(m,1H),3.52(m,1H),2.35(m,2H),2.07 -1.94(m,2H),1.58(m,2H),1.30-1.26(m,28H),0.89(m,3H).
  • compound 11 was prepared from cordycepin through the steps of protection, acylation, and deprotection, in which stearanoyl chloride was replaced by cyclopentylformyl chloride, and the overall yield was 72%.
  • compound 12 was prepared from cordycepin through the steps of protection, acylation, and deprotection, in which stearanoyl chloride was replaced by pyridine-3-carboxylic acid chloride, and the overall yield was 76%.
  • the detection results of the prepared compound 12 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 11.05 (s, 1H), 8.86 (d, 2H), 8.41 (s, 1H), 8.22 (s, 1H), 8.01(d,2H),6.15(d,1H),5.22(d,1H),4.98(m,1H),4.02(m,1H),3.75(m,1H),3.58-3.46(m,2H) ,2.08-1.88(m,2H).
  • reaction mixture was poured into a cold mixture of H 2 O (50 mL), saturated NaHCO 3 (35 mL) and CH 2 Cl 2 :Et 2 O (1:2, 30 mL) for extraction. Extract twice with CH 2 Cl 2 :Et 2 O (1:2, 30 mL). The organic extracts were washed with brine, dried over anhydrous Na2SO4 , and dried under vacuum (keeping the temperature below 40°C).
  • the prepared compound 13b was prepared according to the phosphorylation method in Example 2 and the deprotection method in Example 1 to prepare compound 13, with a yield of 65%.
  • the detection results of the prepared compound 13 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.38 (s, 1H), 6.98 (s, 2H), 6.16 (d, 1H), 5.36 (d, 1H), 4.28(m,1H),4.21(s,2H),4.02(m,1H),3.95(m,1H),3.74(m,1H),2.02-1.82(m,2H).
  • the prepared compound 14d was prepared according to the phosphorylation method in Example 2 and the deprotection method in Example 1 to prepare compound 14, with a yield of 71%.
  • the detection results of the prepared compound 14 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 12.22(s,1H),8.36(s,1H),6.99(s,2H),6.16(d,1H), 5.33(d,1H),4.30(m,1H),4.21(s,2H),4.02(m,1H),3.94(m,1H),3.71(m,1H),2.06-1.88(m,2H) .
  • the prepared compound 15c was prepared according to the phosphorylation method in Example 2 and the deprotection method in Example 1 to prepare compound 15, with a yield of 77%.
  • the detection results of the prepared compound 15 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.58 (s, 1H), 8.37 (s, 1H), 7.07 (s, 2H), 6.15(d,1H),5.33(d,1H),4.70(m,1H),4.44(m,1H),4.28(m,1H),4.18(s,2H),4.02(m,1H)3.55( m,1H). 13 C NMR (100MHz, DMSO-d6) ⁇ 155.3,151.4,148.9,141.1,119.6,97.2,90.1,79.0,73.4,61.5.MSI-MS:350.2[M+H] + .
  • Compound 7 was prepared according to the method of Example 7, and compound 16 was prepared using compound 7 as raw material. Take 5.34g (10mmol) of compound 7, dissolve it in 100mL of dichloromethane solution, and then add 1.3mL of trifluoromethanesulfonic acid ( 1.50g, 10mmol). After the reaction was stirred for 10 minutes, 2.7 mL of trimethylsilyl triflate (10 mmol) was slowly added dropwise to the solution, and the resulting mixture was stirred at -40°C for 30 min. Then 3.96 g (40 mmol) of trimethylnitrilesilane was slowly added, and the mixture was stirred for 2 h.
  • the detection results of the prepared compound 16 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.57(s,1H),8.36(s,1H),7.41(m,2H),7.22(m,3H), 7.09(s,2H),6.17(d,1H),4.99(d,1H),3.97(m,1H),3.74-3.33(m,6H),2.88(m,1H),2.04-1.92(m, 2H),1.28-1.14(d,9H).
  • Compound 13b was prepared according to the method of Example 13.
  • Compound 17a was prepared using 13b as raw material according to the protection method of 1b in Example 1.
  • Compound 17 was prepared using compound 17a as raw material according to the method of preparing 3a in Example 3, wherein: The equivalent of diethyl toluylsulfoxymethylphosphonate is replaced by (((tolyloxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropyl) acid), replacing equivalents of 1b with 17a, the overall yield was 61%.
  • the detection results of the prepared compound 17 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.33 (s, 1H), 6.99 (s, 2H), 6.88 (s, 4H), 6.16 (d, 1H), 5.37(d,1H),4.02(m,1H),3.95(m,1H),3.85(s,2H),3.61-3.42(m,2H),2.05-1.88(m,2H),1.25(s, 18H).
  • Example 18 ((((((5-(6-amino-9H-purin-9-yl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphoryl)bis( Oxy))bis(methylene)bis(2,2-dimethylpropionic acid)
  • Compound 18a was prepared by referring to the method of Example 15 and the protection method of 1b in Example 1.
  • Compound 18a was prepared by using compound 18a as raw material and referring to the method of preparing 3a in Example 3, wherein p-toluoylsulfoxymethylphosphonic acid di Replace equivalents of ethyl ester with (((tolyloxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionic acid) and equivalents of 1b with 18a, the overall yield was 34%.
  • the detection results of the prepared compound 18 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.59 (s, 1H), 8.32 (s, 1H), 7.09 (s, 2H), 6.84 (s, 4H), 6.15(d,1H),5.35(d,1H),4.71-4.64(m,2H),3.85(m,2H),3.60-3.33(m,3H),1.27(s,18H). 13 C NMR( 100MHz, DMSO-d6) ⁇ 175.9,157.3,151.5,149.8,141.3,118.9,98.2,92.8,91.5,79.4,73.3,71.4,70.5,38.6,27.8.MSI-MS:592.4[M+H] + .
  • Compound 14d was prepared by referring to the method of Example 14.
  • Compound 19a was prepared using 14d as raw material according to the protection method of 1b in Example 1.
  • Compound 19 was prepared by using compound 19a as raw material and referring to the method of preparing 3a in Example 3.
  • p-toluene The equivalent of diethyl acylsulfoxymethylphosphonate is replaced by (((tolyloxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2,2-dimethylpropionic acid) ), replacing the equivalent of 1b with 19a, the overall yield was 18%.
  • the detection results of the prepared compound 19 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 12.12(s,1H),8.34(s,1H),7.01(s,2H),6.84(s,4H), 6.12(d,1H),5.35(d,1H),4.01(m,1H),3.93(m,1H),3.85(s,2H),3.63-3.37(m,2H),2.06-1.85(m, 2H),1.27(s,18H).
  • Compound 17a was prepared according to the method of Example 13 and Example 17, and compound 20 was prepared according to the method of Example 7, in which the equivalent of diethyl p-toluene sulfoxymethylphosphonate was replaced by isopropyl (phenoxy) ((Tolyloxy)methyl)phosphoryl)alanine ester, 1b replaced by 17a, overall yield 67%.
  • the detection results of the prepared compound 20 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.37(s,1H),7.40(m,2H),7.20(m,3H),7.02(s,2H),6.15(d,1H),5.38(s,1H),4.94(m,1H) 13 C NMR (100MHz, DMSO-D6) ⁇ 172.1,157.3,156.8,150.0,140.2,121.3,120.6,119.2,76.6,75.0,72.1,69.4,34.5,21.7,19.5. MSI-MS :553.5[M+H] + .
  • Compound 13b was prepared by referring to the method of Example 13.
  • Compound 13b was prepared by referring to the method of preparing 10b from 10a in Example 10.
  • Compound 21a was prepared by referring to the method of preparing 10b from 10a in Example 10.
  • Compound 21 was prepared by referring to the deprotection method of 1a in Example 1. Total The yield is 74%.
  • the detection results of the prepared compound 21 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 10.53(s,1H),8.34(s,1H),6.15(d,1H),5.6(d,1H), 5.02(m,1H),4.01(m,1H),3.78(m,1H),3.57(m,1H),3.51(m,1H),2.35(m,2H),2.09-1.91(m,2H) ,1.52(m,2H),1.33-1.25(m,28H),0.88(m,3H).
  • adenosine as raw material, prepare 15b according to the method in Example 15. Using 15b as raw material, dissolve 6.28g (10mmol) of 15b in ethyl acetate (40mL) under nitrogen protection, and add 37% hydrochloric acid triethylamine solution. 10 mL (22 mmol), stir and heat to 70°C for about 8 hours.
  • compound 22 was prepared according to the method of preparing 10b from 10a in Example 10, combined with the deprotection method of 1a in Example 1, with an overall yield of 79%.
  • the detection results of the prepared compound 22 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 10.60 (s, 1H), 8.67 (s, 1H), 8.35 (s, 1H), 6.15 (d, 1H), 5.33(d,1H),4.98(m,1H),4.31(m,1H),4.02(m,1H),3.78(m,1H),3.58(m,1H),3.53(m,1H),2.32 (m,2H),1.51(m,2H),1.34-1.21(m,28H),0.87(m,3H).
  • Compound 12 was used as a raw material, and compound 23a was prepared according to the protection method of 1b in Example 1.
  • Compound 23 was prepared using 23a as a raw material, and compound 23 was prepared according to the process of preparing 17 from 17a in Example 17. The equivalent of 17a in the reaction was replaced by 23a. The total reaction was The yield was 81%.
  • the detection results of the prepared compound 23 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 11.05, (s, 1H), 8, 85 (d, 2H), 8.34 (s, 1H), 8.18 (s, 1H),8.00(d,2H),6.84(s,4H),6.14(d,1H),5.35(d,1H),4.01(m,1H),3.94(m,1H),3.86(s,2H ),3.60-3.38(m,2H),2.06-1.84(m,2H),1.27(s,18H).
  • Compound 23a was prepared according to the method in Example 23, and compound 24 was prepared according to the method in Example 7 using 23a as raw material, in which the equivalent of compound 1b was replaced by 23a, and the overall yield was 85%.
  • the detection results of the prepared compound 24 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 11.08(s,1H),8.81(d,2H),8.36(s,1H),8.18(s,1H), 8.02(d,2H),7.42(m,2H),7.21(m,3H),6.16(d,1H),5.35(s,1H),4.92(m,1H),4.01-3.95(m,2H) ,3.65-3.54(m,5H),3.35(m,1H),2.04-1.84(m,2H),1.25-1.18(d,9H).
  • Adenosine 2.67g (10.0mmol) and PPh 3 5.78g (22mmol) were added to the round-bottomed flask under an argon atmosphere. Then DMF 18 mL was added and the resulting mixture was stirred at room temperature until the solid was completely dissolved. The solution was cooled to 10°C, and 4.4 mL (2.2 mmol) of diisopropyl azodicarboxylate (DIAD) was quickly added dropwise. The reaction mixture was allowed to reach room temperature. After stirring at 40°C for 2 hours, DMF was evaporated under reduced pressure to obtain a viscous liquid and the mixture was partitioned between 150 mL of water and 100 mL of diethyl ether.
  • DIAD diisopropyl azodicarboxylate
  • test results of the prepared compound are as follows: Bottom: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.57(s,1H),8.36(s,1H),7.10(s,2H),6.37(d,1H),4.59(m,1H),3.95 (s,1H),3.57-3.50(m,2H),2.62-2.55(m,2H). 13 C NMR(100MHz,DMSO-d6) ⁇ 156.3,152.5,149.9,140.5,119.5,90.4,82.6,63.5, 60.8,59.6.MSI-MS:250.2[M+H] + .
  • Compound 25c was prepared according to the preparation method in Example 25, compound 26a was prepared by using 25c as raw material according to the method for preparing compound 17 from compound 17a in Example 17, and compound 26 was prepared by the deprotection method of 1b in Example 1, Among them, 25c was equivalently replaced by 17a, and the overall yield was 54%.
  • the detection results of the prepared compound 26 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.58 (s, 1H), 8.34 (s, 1H), 7.04 (s, 2H), 6.82 (s, 4H), 6.15(d,1H),5.35(d,1H),4.00(m,2H),3.85(s,2H),3.62-3.41(m,2H),1.84(m,1H),1.26(s,18H) .
  • Compound 25c was prepared according to the preparation method in Example 25. Using 25c as the raw material, according to the preparation method in Example 7, the equivalent of 1b was replaced by 25c to prepare compound 27. The total yield was 43%.
  • the detection results of the prepared compound 27 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.58 (s, 1H), 8.34 (s, 1H), 7.42 (m, 2H), 7.21 (m, 3H), 7.07(s,2H),6.15(d,1H),5.34(s,1H),4.95(d,1H),3.99(m,2H),3.76-3.34(m,6H),1.88(m,1H) ,1.27-1.16(d,9H).
  • Example 12 Using the prepared compound 27 as a raw material, refer to the protection method of 1a or 1b in Example 1 to prepare compound 28a. Using compound 28a as a raw material, refer to the preparation method of Example 12, replace the equivalent of cordycepin or 1b with the compound 28a, compound 28 was prepared with an overall yield of 37%.
  • the detection results of the prepared compound 28 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 11.07 (s, 1H), 8.83 (d, 2H), 8.35 (s, 1H), 8.20 (s, 1H), 8.01(d,2H),7.41(m,2H),7.21(m,3H),6.15(d,1H),5.35(s,1H),4.94(d,1H),4.00(m,2H),3.72 -3.32(m,6H),1.82(m,1H),1.24-1.17(d,9H).
  • Compound 1c was prepared according to the protection method for preparing 1c in Example 1. Further reaction was carried out using 1c as raw material. Compound 1c 9.95g (27.2mmol) and CbzCl 4.88g (27.2mmol) were added to 38.5mL toluene and 38.5mL water. In addition, 4.70g (34mmol) of K 2 CO 3 was added, and the mixture was stirred vigorously at a temperature below 25°C. After stirring at room temperature for 3 h, 0.275 g (2.72 mmol) of triethylamine and 5.78 g of sodium chloride were added in sequence, and the mixture was stirred for another 30 min. The organic layer was separated and concentrated to obtain the desired oily product 29a, weighing 13.1 g, with a yield of 90%, MSI-MS: 522.6 [M+Na] + .
  • the detection results of the prepared compound 29 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.89(s,2H),8.58(s,1H),8.37(s,1H),7.05(s,2H), 6.73(d,1H),5.01(m,1H),4.92(s,1H),4.27(m,1H),3.75(m,1H),3.56-3.47(m,2H),2.38(m,1H) ,2.13(m,1H),1.89(m,1H),0.98(d,6H).
  • compound 32a was prepared by replacing equivalent amounts of compound 1b with compound 29 according to the preparation method in Example 3, with a yield of 77%.
  • Compound 32a was used as the substrate, and compound 32 was prepared according to the preparation method of Example 12 by replacing the equivalent of 1a with compound 32a, with a yield of 90%.
  • the detection results of the prepared compound 32 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 11.05(s,1H),8.91(s,2H),8.84(d,2H),8.35(s,1H), 8.18(s,1H),8.01(d,2H),6.75(d,1H),5.01(m,1H),4.81(s,2H),4.24(m,1H),3.95(m,1H),3.73 (d,2H),3.60-3.35(m,2H),2.37(m,1H),2.14(m,1H),1.89(m,1H),0.96(d,6H).
  • the detection results of the prepared compound 33 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.58 (s, 1H), 8.33 (s, 1H), 7.42 (m, 2H), 7.21 (m, 3H), 7.08(s,2H),6.15(d,1H),5.00(m,1H),4.02(m,1H),3.71-3.48(m,8H),2.24-1.98(m,2H),1.25(d, 3H).
  • Compound 33a was prepared according to the process of Example 33, using 33a as raw material, and according to the preparation method of Example 12, replacing the equivalent of 1a with compound 33a to prepare compound 34.
  • the total yield was 83%.
  • the detection results of the prepared compound 34 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 11.02(s,1H),8.81(d,2H),8.34(s,1H),8.17(s,1H), 8.02(d,2H),7.41(m,2H),7.23(m,3H),6.16(d,1H),4.98(m,1H),4.01(m,1H),3.78-3.47(m,8H) ,2.25-1.97(m,2H),1.27(d,3H).
  • Compound 17a was prepared according to the method of Example 17. Using 17a as a raw material, it was prepared according to the preparation method of Example 8, in which the equivalent of 2-chloro-3-hydroxypropylvaline was replaced by 3-chloro- 5-(hydroxymethyl)cyclopentane-1,2-diol was used to prepare compound 36a. After deprotection, compound 36 was prepared with a total yield of 41%.
  • the detection results of the prepared compound 36 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.37(s,1H),7.01(s,2H),6.15(d,1H),5.92(s,1H), 5.37(d,1H),4.38(s,1H),4.25(s,1H),4.02(m,2H),3.84(m,1H),3.65-3.31(m,6H),2.07-1.82(m, 2H),1.70-1.44(m,3H).
  • Example 37 Preparation of compound: ((((((5-(6-amino-9H-purin-9-yl)-2-azido-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)phosphorus Acyl)bis(oxy)bis(methylene)bis(2,2-dimethylpropionic acid), its preparation process is as follows:
  • Compound 37 was prepared by using 37b as a raw material according to the method for preparing compound 17 from compound 17a in Example 17, in which 37b was substituted for 17a in equivalent amounts, and the overall yield was 50%.
  • the detection results of the prepared compound 37 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.58 (s, 1H), 8.34 (s, 1H), 7.07 (s, 2H), 6.82 (d, 4H), 6.15(d,1H),5.34(d,1H),4.02(m,1H),3.84(d,2H),3.61-3.42(m,2H),2.11-1.80(m,2H),1.28(d, 18H).
  • Compound 38 was prepared from compound 2 by using compound 2 as raw material and using the method of Example 35. The overall yield was 24%.
  • the detection results of the prepared compound 38 were as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.35 (s,1H),7.09(d,1H),6.95(s,2H),6.68(d,1H),6.15(d,1H),5.35(s,1H),4.27-4.02(m,5H), 3.74(m,1H),2.07-1.84(m,2H). 13 C NMR(100MHz,DMSO-d6) ⁇ 156.1,152.3,149.8,140.2,134.9,124.2,119.5,98.7,74.9,74.3,68.1,34.5. MSI-MS:458.3[M+N] + .
  • Example 39 Preparation of compound: (5-(6-amino-8-(isopropylamino)-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)dihydrogenphosphate, and its preparation The process is as follows:
  • Compound 39 was prepared using compound 1b as raw material. First, 3.65g (10mmol) of compound 1b and 1.18g (20mmol) of 2-propylamine were added to the reaction bottle, and 60mL of dioxane was added as a solvent. The reaction was carried out at 80°C. The reaction was refluxed for 20 h, and the progress of the reaction was monitored by TLC. After the reaction is completed, the reaction solution is concentrated, and then 50 mL of water and 50 mL of ethyl acetate are added for extraction twice. The organic phases are combined, concentrated under reduced pressure to obtain an oily liquid, and purified by column chromatography to obtain a light yellow oily liquid as compound 39a, weighed as 1.44g, yield 34%.
  • Example 40 Preparation of compound: 3-((5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)-5-(hydroxymethyl)cyclopenta Alkane-1,2-diol, its preparation process is as follows:
  • the detection results of the prepared compound 40 are as follows: 1 H NMR (400MHz, DMSO-d6) ⁇ 8.58(s,1H),8.37(s,1H),7.04(s,2H),6.15(d,1H), 5.91(s,1H),5.37(d,1H),4.37(s,1H),4.25(s,1H),4.00(m,2H),3.82(m,1H),3.63-3.32(m,6H) ,2.08-1.81(m,2H),1.71-1.45(m,3H).
  • Example 41 Experimental anti-tumor effect of modified derivatives of cordycepin on liver cancer
  • mice were randomly divided into twelve groups. , 10 animals in each group, respectively a blank group, a cordycepin control group and a drug group (compounds 2, 3, 5, 7, 15, 24, 27, 31, 35, 40); the blank group was given corn syrup by gavage every other day Nutritional supplements and DMSO were used as solvent controls.
  • the cordycepin control group and the drug group were administered the compound drug (500ug/time/animal) by gavage every other day, and were observed continuously for 14 days; the changes in liver cancer tumor size in the two groups of mice were observed. After the experiment, the mice were killed, and blood and corresponding tissue specimens were collected. The tumor tumors were immediately photographed and weighed. Part of the tumor tissue was fixed in formalin solution for further testing. Two transplanted tumors were measured every two days with a caliper. The vertical diameter (length and width), calculated transplanted tumor size, tumor volume change experimental results and tumor results of the blank group, cordycepin control group and drug group within 14 days are shown in Figure 1.
  • tumor volume (mm 3 ) 1/2 ⁇ (length ⁇ width) 2 .
  • the experimental results show that the drug group alone can significantly inhibit the growth of mouse tumors, and the effects are better than the cordycepin control group.
  • compound 24 has excellent anti-liver cancer tumor effect (900mm 3 reduced to ⁇ 100mm 3 ) (P ⁇ 0.001), indicating that the compound The drug group has the effect of killing liver cancer tumor cells or activating tumor immunity.
  • Example 42 Experimental anti-tumor effect of modified derivatives of cordycepin on small cell lung cancer
  • the MTT assay was used to detect the effects of cordycepin, compounds 1,2,4,5,7,8,10,12,16,18,21,23,24,35,38 and cordycepin in small cell lung cancer cell lines H1048 and The anti-tumor dose-effect relationship curves of H446 and H69 were analyzed.
  • the results of calculating the half inhibitory concentration (IC 50 ) are summarized in the table below.
  • In vitro anti-tumor experimental results show that compared with cordycepin, the effective concentration of modified cordycepin derivatives on tumor cells is reduced.
  • compounds 35 and 38 have strong in vitro killing effects on three types of cells, but their toxicity is also larger, followed by compounds 18 and 21.
  • the IC 50 of cordycepin in small cell lung cancer cell lines H1048 and H446 are both higher than 100 ⁇ M, and the effective concentration is relatively high. In practical applications, a large amount of drugs may be needed to be effective.
  • Example 43 Determination of the maximum tolerance of cordycepin modified derivatives to zebrafish in vivo experiments
  • the lethality and developmental malformation of zebrafish at different doses were examined to ensure that the survival rate was >90% and the teratogenic rate was The concentration of ⁇ 20% determines the maximum tolerated dose (MTD) of the test article to normal zebrafish.
  • Cordycepin was selected as the control group, and compound 2 and compound 5 were selected as the others.
  • Compound 6, compound 7, compound 9, compound 11, compound 13, compound 20, compound 24, compound 25, compound 28, compound 35, compound 36, compound 37, compound 39 were tested for MTD of zebrafish according to the above method, and the test results were As shown in table 2.
  • Table 2 Maximum tolerated dose (MTD) of natural products and positive control drugs in zebrafish model
  • Example 44 Inhibitory effect of modified derivatives of cordycepin on small cell lung cancer in zebrafish
  • H69, H446, and H1048 cell zebrafish-bearing small cell lung cancer xenograft tumor models prepare H69, H446, and H1048 cell suspensions at a concentration of 1 ⁇ 10 6 mL and place them in serum-free cell culture medium, per ml. Add 5 mL of red fluorescent dye (CM-DiI) cell labeling solution to the cell suspension, mix gently, incubate at 37°C for 20 minutes, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, and re-add serum-free medium for resuspension. Then repeat the above steps twice to obtain the required labeled cell suspension, which is transplanted into the zebrafish yolk sac by microinjection. About 100 cells are transplanted into each tail to establish zebrafish-human small cell lung cancer sensitive line transplantation. Tumor model, and zebrafish injected with ovarian cancer cells were cultured at 35°C to 3dpf.
  • CM-DiI red fluorescent dye
  • Tumor growth inhibitory effect (%) S (model control group) - S (drug group) / S (model control group) ⁇ 100%
  • mice were randomly divided into seven groups. There are 8 animals in the group, which are respectively the control group IgG, the compound 16 drug + IgG group, the PD-1 antibody treatment group, the CTLA4 monoclonal antibody treatment group, the compound 16 drug + PD-1 combined treatment group, and the compound 16 drug + CTLA4 monoclonal antibody combined treatment group. Group.
  • Control group IgG Immunoglobulin G (IgG) (500ug/time/animal) was intragastrically administered every day for 28 consecutive days;
  • Cordycepin+IgG Immunoglobulin G (IgG) and cordycepin (both were intragastrically administered every day) (500ug/time/animal) for 28 consecutive days;
  • compound 16 + IgG group immunoglobulin G (IgG) and compound 16 (both 500ug/time/animal) were administered intragastrically every day for 28 consecutive days;
  • Compound 16 + antibody combined treatment group Immunoglobulin compound 16 (500ug/time/animal) was intragastrically administered every day , administer the antibody (500ug/time/animal) by gavage every 4 days for 28 consecutive days.
  • mice were killed, and blood and corresponding tissue specimens were collected.
  • the tumor tumors were immediately photographed and weighed. Part of the tumor tissue was fixed in formalin solution for further testing.
  • cordycepin has an anti-tumor growth effect (size 1300mm 3 reduced to 730mm 3 ), and compound 16 alone has a significantly greater anti-tumor growth effect than cordycepin (size 1300mm 3 reduced to 480mm 3 ) ( P ⁇ 0.01)
  • immune checkpoint inhibitors PD-1 and CTLA4 used alone also have significant anti-tumor growth effect (size reduced from 1300mm 3 to about 448mm 3 ) (P ⁇ 0.01), compound 16 and immune checkpoint inhibitor PD
  • the synergistic effect of the two can greatly improve the anti-tumor effect (the size is reduced from 1300mm3 to about 260mm3 ).
  • Example 46 Experimental Inhibitory Effect of Cordycepin Modified Derivatives on Melanoma
  • mice Suspend 2.5 ⁇ 10 B16-F10 melanoma cells in 100 ⁇ L of PBS and inoculate the outer thigh of the left lower limb of C57BL/6j nude mice; after 1 week, the transplanted tumor reaches a size of approximately 100 mm. , the mice were randomly divided into ten groups, with 10 mice in each group, namely, blank control group, cordycepin group, compound 13 group, compound 14 group, compound 17 group, compound 19 group, compound 22 group, compound 26 group, compound 29 groups and 34 compounds.
  • the blank control group corn steep liquor and other nutrients and DMSO were administered by gavage every day as the solvent control group for 24 consecutive days; the compound group: the corresponding compound (both 500ug/time/animal) was administered by gavage every other day for 24 consecutive days. ;
  • the size changes of melanoma tumors in the ten groups of mice were observed every 4 days in the early stage and every two days in the later stage. After the experiment, the mice were sacrificed, and blood and corresponding tissue samples were collected. The tumor tumors were immediately photographed and weighed, and part of the tumor tissue was taken and fixed.
  • Example 46-1 Compound 27 was used to inhibit melanoma in the above manner, and it had the same level of inhibitory effect as Compound 26.
  • Example 47 Experimental Inhibitory Effect of Cordycepin Modified Derivatives on Ovarian Cancer
  • mice Suspend 2.5 ⁇ 10 ID8 ovarian cancer cells in 100 ⁇ L of PBS and inoculate C57BL/6j nude mice on the outside of the left lower limb of the thigh; about one week later, when the transplanted tumors reach a size of approximately 100 mm, the mice are randomly divided into five groups, each group contains 10 Only, they are the control group, cordycepin group, compound 24 drug group, PD-1 + TIM3 antibody treatment group, and compound 24 drug + PD-1 + TIM3 antibody treatment group.
  • Control group Immunoglobulin G (IgG) (500ug/time/animal) and PBS were administered by gavage every day for 15 consecutive days;
  • Cordycepin group Cordycepin (500ug/time/animal) was administered by gavage every day together with PBS.
  • Compound 24 drug group Compound 24 (500ug/time/animal) was administered intragastrically every day and mixed with PBS for 15 consecutive days;
  • PD-1+TIM3 antibody treatment group every 4 days The antibody PD-1+TIM3 (500ug/time/animal respectively) was administered by gavage for 15 consecutive days;
  • the compound 24 drug + PD-1+TIM3 antibody treatment group the immunoglobulin compound 24 drug (500ug/animal) was administered by gavage every day. times/animal), and the antibody PD-1+TIM3 (500ug/time/animal respectively) was intragastrically administered every 4 days for 15 consecutive days.
  • the size changes of ovarian tumors in the five groups of mice were observed every two days.
  • mice were sacrificed, and blood and corresponding tissue samples were collected.
  • the experimental results are shown in Figure 5.
  • Experimental results show that cordycepin has an anti-proliferation effect on ovarian cancer (the size is reduced by 1.5 times at 15 days).
  • Compound 24 alone has a significantly greater anti-tumor growth effect than cordycepin (the size is reduced by about 2 times at 15 days).
  • Immune checkpoint inhibition When the agents PD-1 and CTLA4 are used alone, they also have significant anti-tumor growth effects (the size is reduced by more than 2 times in 15 days). When compound 24 and the immune checkpoint inhibitors PD-1 and CTLA4 are used simultaneously, they can greatly increase the anti-tumor growth effect. Effect of tumors (approximately 4-fold reduction in size at 15 days).
  • Example 48 Experimental inhibitory effect of modified derivatives of cordycepin on gastric cancer cells in vitro
  • the MTT assay was used to analyze the anti-tumor dose-effect relationship curves of cordycepin and forty prepared compounds in gastric cancer cell lines AGS and BGC-823 respectively.
  • the results of calculating the half inhibitory concentration (IC 50 ) are summarized in Table 4 below.
  • In vitro anti-tumor experimental results show that compared with cordycepin, the effective concentration of modified cordycepin derivatives on tumor cells is reduced.
  • Example 49 Experimental effect of cordycepin modified derivatives on in vivo tumor inhibition of gastric cancer-mouse model
  • mice Suspend 2 ⁇ 10 7 BGC-823 gastric cancer cells in 100 ⁇ L of PBS and inoculate C57BL/6j nude mice.
  • the anterior chest wall is disinfected with 75% ethanol, and the most obvious apical pulse is touched with hand, about Inoculate into the left ventricle in the second intercostal space 3 mm to the left of the sternum.
  • the mice are randomly divided into five groups, with 10 mice in each group, namely the control group and the cordycepin group. Compounds 16, 18, 24 drug group.
  • Control group Corn steep liquor and other nutrients and DMSO were administered by gavage every day as the solvent control group.
  • Cordycepin group Cordycepin (500ug/time/animal) was administered by gavage every day and mixed with DMSO;
  • Compound drug group Administration was administered by gavage every day.
  • Compounds (500ug/time/only) were mixed with DMSO.
  • the modified cordycepin has a more obvious effect than cordycepin.
  • the compound 24 group was reduced by about 12 times (from 1.73g to 0.14g) compared with the control group.
  • Example 50 In vivo tumor inhibitory effect of modified derivatives of cordycepin on pancreatic cancer-mouse model
  • Pancreatic cancer Pan02-luc cells were placed in DMEM medium containing 10% fetal calf serum in a 37°C CO 2 incubator. Trypsin ED-TA digestion and passage were performed every 2 to 3 days. When the cells reach the required number, take the cells in the logarithmic growth phase and resuspend them in culture medium to 1 ⁇ 10 7 /mL. C57BL/6j nude mice were fed under pathogen-free conditions. When the mice grew to 6 weeks, 200uL pancreatic cancer Pan02-uc line cells were subcutaneously injected into the right dorsal near axilla of each mouse. The transplanted tumors appeared within about a week.
  • mice were randomly divided into five groups, with 10 mice in each group, namely the control group, the cordycepin group, and the compound 11, 16, and 24 drug groups.
  • Control group Corn steep liquor and other nutrients and DMSO were administered by gavage every day as the solvent control group.
  • Cordycepin group Cordycepin (500ug/time/animal) was administered by gavage every day and mixed with DMSO;
  • Compound drug group Administration was administered by gavage every day.
  • Compounds (500ug/time/only) were mixed with DMSO. Administer continuously for 24 days. Afterwards, the survival rates of the five groups of mice were observed every day to study the median survival time. The experimental results are shown in Figure 7.

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  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un composé dérivé de cordycépine ayant un effet antitumoral, qui a une structure telle que représentée par la formule I, le dérivé de cordycépine et une composition pharmaceutique de celui-ci selon la présente invention ont un bon effet contre la prolifération tumorale. Par comparaison avec un médicament parent, le dérivé de cordycépine a une meilleure affinité pour une membrane cellulaire, de telle sorte que le médicament a une demi-vie plus longue du métabolisme in vivo, et le temps de rétention dans le corps est plus long. Par comparaison avec d'autres médicaments nucléosidiques anti-tumoraux, le dérivé de cordycépine et la composition pharmaceutique de celui-ci selon la présente invention ont un effet sur une plus grande plage de tumeurs et ont également des effets plus larges sur les tumeurs. En particulier, le dérivé de cordycépine et la composition pharmaceutique de celui-ci selon la présente invention ont de bons effets d'inhibition sur le cancer gastrique, le cancer du pancréas, le cancer du foie, le cancer du poumon à petites cellules, le cancer colorectal, le mélanome, le cancer de l'ovaire, etc, et ont des effets secondaires inférieurs et une meilleure efficacité.
PCT/CN2023/099859 2022-07-12 2023-06-13 Composé dérivé de cordycépine ayant un effet antitumoral WO2024012126A1 (fr)

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CN116478225A (zh) * 2023-04-12 2023-07-25 南京工业大学 一种丝氨酸改性的虫草素磷酸酯药物分子的制备方法与应用

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